All cells must sense their environmental conditions and appropriately respond to conditions in order to survive. The upstream components of the inorganic phosphate signal transduction pathway (PHO pathway) in budding yeast will be investigated as a paradigm of how cells sense and respond to extracellular stimuli. The regulation of the PHO pathway is relatively well understood downstream of Pho80-Pho85-Pho81, a cyclin, cyclin-dependent kinase (CDK) and CDK inhibitor complex. However, little is known about how extracellular phosphate concentrations are sensed and how this information is transmitted to the CDK complex. The aims of this project are: (1) To define the role of the phosphate transport system in signaling extracellular phosphate concentrations by inactivating all known (and putative) phosphate transporters and assaying their effect on the PHO pathway, phosphate uptake and viability. (2) To identify factors that regulate CDK activity utilizing a genetic selection for mutants unable to induce phosphate-responsive genes. (3) To identify covalent modifications to the CDK complex that are relevant to the regulation of CDK activity using mass spectrometry. These studies should identify factors required for the regulation of CDK activity. Pho85, shares significant sequence similarity with mammalian Cdk5, and Pho81 shares sequence similarity with p16(INK4), which is encoded by a tumor suppressor gene in humans. Studies of the PHO pathway may allow for a better understanding of how important proteins, CDKs and CDK inhibitors, in human cells are regulated.